1. Field of the Invention:
The present invention relates to an improvement in the field of cytology and more particularly a microscopic method of supravital blood analysis by which under normal white light illumination of a microscopic field optical differentiation, identification, comparisons and enumeration of each one of a series of leukocytes found in normal and pathologic human blood is made possible by use of a single pure dye without fixation and more accurately and rapidly than heretofore by either manual or automated differential leukocyte counters.
2. Description of the Prior Art
Ehrlich made biological elements more readily and easily recognized under microscopic examination and for photographic observation by use of dye stains (aniline dyes) to identify certain white blood cells. Ehrlich was the first to note that some dyes were metachromatic, observing that the staining of the cell or components such as granules of leukocytes causes the cell to take on a color different than that of the stain in solution or expected color from the stain. Basophils, for example, were observed to take on a color different from the stain. Other histological specimens other than blood cells have also been reported to stain in a plurality of identifiably different colors.
A review of the state of the art indicates it is almost universal practice, before staining (which presently uses a plurality of chemical differing dyestuffs in admixture) to employ a fixative procedure which may require up to an half hour treatment before the biological specimen is subjected to dye stain. Fixatives are generally preservatives and denaturants that often interfere with the sensitivity of the dye sorption. Illustratively, fixatives include formaldehyde both as liquid and vapor, absolute alcohols (methyl), picroformal, etc. Very often living cells do not stain using vital dyes and fixatives have been essential to staining the specimens. Cytochemistry includes considerable information on techniques developed to assure reproducible staining of blood cells. Many essential additives are normally unstable and deteriorate rapidly, thus making cellular identification difficult and in some instances unreliable. Dr. Thomas E. Necheles has observed in relation to leukocyte analysis that this "system has undergone little or no change in fifty years."
Dye staining does serve, however, as a means of discernment of otherwise undiscernable detail of conferring a color reaction on cells and their stainable components; metabolic, functional or pathological.
United States hospitals began leukocyte counting in the early 1900's, using the count as indicia as to whether emergency surgery was necessary, for example. In the U.S. alone, more than half a million differential counts are performed every day, most of them by manual methods. It is important that total white cell counts and differential cell counts be performed and reported without delay. Time is of essence and providing required analysis more rapidly is a desideratum.
The value of leukocyte counting having been established, the demand for rapid blood analysis has developed so that beginning about 1950 with the work of Mellors and Papincolaou (1952) development of automated differential leukocyte counting instrumentation means had developed into a plurality of instruments by 1980. The CYDAK unit was early used to investigate the feasability of blood cell classification which pointed up the importance of specialized staining procedures and features were extracted from optical density histograms of each cell image. The procedure established that cells could be differentiated into four of the five classes of leukocytes, namely; neutrophils, eosinophils, lymphocytes and monocytes. Young (1969) published results on an automated classification of five cell classes and Bacus in 1971 extended the differentiation.
However, it is understood that automated differential systems presently rely upon multiple dye usage and dye degradation systems or indirect fluorescent mesaurement using fluorescent dyes.
In the prior art staining of blood it has been observed that it is practice to use two or more stains in combination (Romanowski, Giemsa and Wright stains). These methods are difficult in practice to provide quality control. The methods require standardization in preparation of each dye stain component as well as in the method of specimen staining. In development of successful automated leukocyte counters, reproducibility of staining is even more important to verifiable analysis.
LARC stainer (used in commercial automated differential leukocyte counter) is reported (Mogler 1973) to be a mixture of some ten thiazine dyes, eosin Y and 2.sup.1, 4.sup.1, 5.sup.1 tribromofluorescein (P. N. Marshall). Present art stains most often are in fixative alcoholic solutions and employ two or more stains in combination. Accurate analysis of vital blood staining is made most difficult. With the difficulty presented in the controlled oxidation of methylene blue essential to Romanowski stains, for example, the problems of quality control of the added ten individually different dye stains as are used in combination become awesome.
It has been recognized in the art that the widespread standardization and adoption of a limited number of stains would ensure greater accuracy and reproducibility in cytological studies. Serious introduction of artifacts have been observed by use of fixatives and cause difficulty in interpretation and misinterpretation in leukocyte differentiation and enumeration. pH adjustments, heavy metal cations have been reported to prevent cytochemical tests from working in the expected manner. Some dyes, particularly azo dyes, are noted to demonstrate non-specific precipitation around cells; other degenerative changes in fixed blood samples include vacuoles, clover-leafing of nuclei, distortion cell shapes and smudges and interference with ideal staining. The importance of performing differential counts on as near living cells in the shortest possible time in order to obtain optimally useful and valuable blood cell analyses has been recognized. Alcoholic dye solutions interfere with supravital staining. So far as is known, freshly prepared water soluble stains exhibit a minimum denaturant effect upon supravital blood during examination. All dyestuffs are more or less toxic to the blood cells, but some are more so than others. It is material that the cells under examination remain living as long as possible. Rapidity of staining obviously shortens the exposure time, thus allowing greater opportunity to examine leukocyte cells before all vitality is lost. Automated differential leukocyte counting in less minutes is sought for.
Studies and review of the prior art of performing microscopic blood analyses and disease diagnosis has indicated it is not unusual for pathologists to warm the dye and the blood specimen to body temperatures (about 37.degree. C.) before contact. Dr. Sabin had a "warm box" to insure temperature control.
It has also been noted that some dyes used in the prior art are quite temperature sensitive. The literature reports that cresylecht violet is not an operative stain above 30.degree. C. It is considered important for the purpose of this method as disclosed herein that the dyestuff be useful to stain leukocytes at temperatures as high as 37.degree. C. and no difficulty has been observed with the select dyes to temperatures of about 40.degree. C.
In the parent application a relatively small number of metachromatic dyestuffs are disclosed as useful in identification of one or more species of leukocyte. Identification and differentiation was specifically related to polymophonuclear leukocytes (neutrophils), basophils, lymphocytes generally, and monocytes. A uniting commonality observed was that all of the dyes found to be operative for the purposes of the parent application metachromatically stained monocytes differentially from others in the above group.
The unusual qualities of the dye basic orange 21 (CI #48035) were observed in relation to the eosinophils, basophils, and monocytes, but as the B-cells are few in number they were intially overlooked. It was initially observed in the parent case that optical differentiation between mature and immature neutrophils appeared potential in that the mature granules were different in chroma from the immature granules which were more red and orange in comparison. As this group, including myeloblasts, promyelocytes, myelocytes, metamylocytes and bands are not always present in all blood specimens or present in significant numbers as is often the case with T-lymphocytes (or T-cells) and B-lymphocytes (or B-cells) they were not then all specifically identified as being metachromatically and differentially stained by basic orange 21.
Subsequent to completion of the work supportive of the parent application, continuing research on the use of this unique dye in similar blood donor studies established that it was reproducibly possible, using this selected basic cationic dye of the methine, polymethine and quinoline class to distinguish through metachromatic response certain lymphocytes. It is also possible further to identify at least ten recognized granulocytes and lymphocytic cells established in the art to be of vital interest to the health sciences.
Further, this differentiation was immediate, it required no complex biochemistry or arduous pre-treatment of the blood specimens. Additionally it was noted the dye exhibited minimum toxicity.
Micro spectrophotometric measurements were made with an aperture small enough to measure the color in the granules of supravitally stained leukocyte granulocytic cells. No other part of the cell entered into the measurements to any extent were found to provide extinction coefficients of the colors of the different leukocyte species which were consistently different and were often of an order of differences in hue, value or chroma of the order of 50 nanometers. These were recognizable peaks, consistent over many cells. It is understood that differences of the order of 5 nm are significant in microspectrophotometric measurements if the differences are consistent and reproducible.
Among the immature granulocytic cells immediately identifiable and distinguishable one from the other are myeloblasts and cells of the myeloid series, namely; promyelocytes, myelocytes and metamyelocytes. These are believed to be and are generally understood to be precursors of the polymorphonuclear leukocytes or neutrophils, which are also stained metachromatically so as to be readily and easily distinguished, identified and enumerated by the supravital blood analyses made possible by the advances disclosed herein.
As disclosed in the parent application, it is also practical at the same time to distinguish neutrophils, eosinophils, basophils, lymphocytes and monocytes from each other and from the foregoing precursors should they all be present in a specific blood sample under microspectrophotometric analyses.
Additionally, it has also been found that this unique dye provides an optically different pattern of color as well as a different density of each color of granule in band-lymphocytes. Thus, this quality of leukocyte cell can also be uniquely separated by optical differentiation from the other immature cells identified above. The differentiations in color, color arrangement and color density are also of such a degree of magnitude of difference that human counting of all the above individually named cells can be done by a competent operator. Evidence available also indicates automatic differential counting equipment will develop based upon and to be accommated by differences due to the presence or absence of color and the physical patterns established in the nucleus and by the relative number, size, arrangement or pattern and hue, value and chroma (color) and color density due to the number of granules in the cytoplasm.
Almost unbelieveably, but also demonstrated in the basic research thus far completed, is the further ability to differentiate B-lymphocytes or B-cells from T-lymphocytes or T-cells. Again, it is possible to specularly identify each of these important lymphocytes, one from the other, qualitatively and quantitatively using the same dyestuff in the same supravital, fixative free analysis as well as to distinguish and enumerate the T-cells and B-cells from each of the foregoing individual immature and mature cells including bands.
As has been disclosed in the parent application, the monocytes also respond metachromatically and are likewise identifiable and countable as there provided without conflict with the foregoing microscopic analyses of the lymphocytes as described.
Mention should also be made that blood platelets, small distinct, dust-like particles that serve one function of control of bleeding can be identified and counted by their orange staining. Their number is also a valuable indicia of blood quality.
Further discovery of the capacity of basic orange 21 to differentiate additionally myeloblasts and blood cells of the myeloid series as well as bands and T-lymphocytes and B-lymphocytes extends the original potential field of usefulness of the dye unexpectedly beyond the capacity recognized in the parent application. Supravital blood specimen fractions of fluids associated with healthy tissue or tissue suspected of abnormality such as plasma, lymph, serum, etc., containing one or more of the above cells after metachromatic staining may be examined microscopically to differentiate each species of cell indicated above permitting enumeration and comparative study.
The present advance in the art, coupled with the parent disclosure establishes unparalleled advance in hematology, cytology and immunology and the ability to plan and conduct researches in an unlimited area of human health. Need for costly reactants, invaluable research time and more accurate data assembly have been thereby measurably advanced.
The art of diagnosis of disease has a new horizon beyond the present limits.
Up to the present writing nearly a thousand different known dyes, some of which are no longer manufactured and were available purely by reason of extensive searches have also been investigated in an attempt to find leads on useful dyes for the broad purpose of supravital blood specimen fraction analyses and study. Trails of innumerable classes of dyes both from chemical structure and chromophoric group classification have been frustrating as to theory, metachromasia being very rarely found, however not exclusively in any known classification. It has been noted, however, that the basic quaternary cationic chemical class has produced more interest than any other group presently known.
In the studies basic to this application, twenty (20) methine and polymethine dyes of basic red, basic orange and basic violet hues were tested for leukocyte identification purposes. In the first group of nine basic reds only four caused instant staining of the nucleus of monocytes, but little or no staining of other cell types. This series includes basic red 13 of the parent application and basic red 35, 36 and 49 since investigated. Of the six (6) basic violet methine and polymethine dyes in the tests, basic violets 7, 15, 16 (disclosed in the parent application), 39 and 40 also instantly and selectively stained monocytes.
Six basic orange polymethine dyes were also subjected to practical testing. Basic orange 21 was unique in this series, not only staining monocytes distinctively, but an unexpected number of other leukocytes ecah were instantly stained, each in a differential way.
Significantly, the 20 polymethine and methine dyes represent the current world inventory of all available as well as commercially obsolescent dyes of the methaine and polymethine classification found in my researches of the prior dye art. The parent application reports a study of eighteen carbocyanine dyes, only one of which displayed similar metachromatic staining of monocytes. Carbocyanines, quinoids and methine and polymethine dyes are often of very similar chemical structure.
Detailed and specific study of the structure of basic orange 21 (Color Index 48035) disclosed in U.S. Pat. No. 2,126,852 led to testing of basic orange 22 (Color Index #48040). A study of the two chemical structures (as reproduced below along with actual testing led to astonishing results. Basic orange 22, does not exhibit metachromasia to any practical level of usefulness for the purposes herein. ##STR1##
Study of these structures indicates the only differences to be observed are that the indolyl radical of each basic orange indicated as B and B.sup.1 in the foregoing structures varies only by a change in the methyl group from a 2 position in basic orange 21 to a 1 position in basic orange 22 and being substituted on a carbon in basic orange 21 and on a N in basic orange 22. The open 2 position in basic orange 22 also has an additional phenyl group in place of the methyl group so re-positioned in the structure.
In over three years of research with wide varieties and chemical classes of dyes no basis for predictability of metachromasia has yet been observed. More frequent metachromasia has been found among the basic cationic quaternary dyestuffs and relatively more often among the methines and polymethines.
Prior art references indicate that it was not unusual in supravital analyses to employ three concentrations of dye in three preparations of slides in such analyses as are an essential check on results. With basic orange 21, the color differentials are so separated and the colors so exceptionally vivid that one can readily distinguish primary from secondary granules, instantly, with one dye and one slide.